CN103848906A - Rice high-temperature-resistant related gene OsZFP, selection marker and separating method of related gene - Google Patents

Abstract

The invention discloses a rice high-temperature-resistant related gene OsZFP, a selection marker and a separating method of the related gene. The invention provides a separated rice endogenous high-temperature-resistant gene (OsZFP gene for short) and a coded polypeptide thereof, a preferred rice cell comprising the high-temperature-resistant gene or the coded polypeptide thereof and a preparation method of a plant cell. The invention further provides a novel method and a novel technology for breeding a high-temperature-resistant new crop variety, including a regulatory sequence relevant to high temperature resistance, a tightly-linked molecular marker for marking the high-temperature-resistant gene and a sequence of the molecular marker.

Description

Rice high temperature resistance related gene OsZFP, screening marker and isolation method thereof

technical field

The invention relates to the technical fields of plant genetic engineering and molecular breeding. Specifically, it relates to the mapping, isolation, functional analysis, sequence characterization, regulatory sequence identification, and co-segregation molecular marker screening and creation of plant high temperature resistance genes and their application in improving the stress resistance of rice and other crops. A method and program for genetic transformation and molecular marker-assisted selection of a heat-resistant gene in rice. In addition, the present invention also relates to the application of improving the stress resistance of crops such as rice by using the high temperature resistance gene. the

Background technique

Rice is the most important food crop and the main source of food for more than half of the world's population (Khush, 2005). According to reports, rice production can only meet people's living needs by 2050 at an annual growth rate of 0.6-0.9% (Carriger and Vallee, 2007). The increase of crop yield depends on farmland, and the sustainable development of agricultural production needs to avoid the deterioration of the environment, the destruction of ecological balance and the loss of biodiversity (Cassman, 1999; Tilman et al., 2002). Crop yield is mainly determined by photosynthesis and respiration, however, both photosynthesis and respiration are sensitive to temperature (Yoshida, 1981), and are also affected by the concentration of CO2 in the air (Baker et al., 1990), the ozone layer (Maggs and Ashmore, 1998), these factors are also related to the greenhouse effect (Rosenzweig and Parry, 1994). the

Continued warming of the climate has had a disastrous effect on rice production. In 2006 and 2007, the provinces of Chongqing, Hubei, Hunan, Anhui, Zhejiang and Guangdong in the middle and lower reaches of the Yangtze River in China experienced large-scale and sustained high-temperature weather above 38°C for two consecutive years, resulting in large-scale production reductions of rice and other food crops, and serious damage to the population. Its seed setting rate is less than 50%. Therefore, there is an urgent need for available high-temperature resistant genetic resources in breeding to cope with the serious threat to rice production brought about by global warming. Therefore, it is undoubtedly of great significance in theory and practice to carry out extensive research on heat tolerance, deeply excavate high temperature resistant genetic resources, and strive to cope with the serious threat of global warming to rice production from the perspective of breeding new heat resistant plant varieties. the

In recent years, rice booting stage (Zhao Zhigang et al., 2006), heading stage (Zhang Yongsheng et al., 2009; Zhang et al., 2009), flowering stage (Cao Liyong et al., 2003, 2002; Pan Yi et al., 2005; Chen Qingquan et al. , 2008; Zhang Tao et al., 2008; Kui Limei et al., 2008; Zhang et al., 2009; Jagadish et al., 2010;), grain filling stage (Zhu Changlan et al., 2005) and rice amylose content synthesis and gel consistency formation (Zhu Changlan et al., 2006) carried out a large number of genetic analyzes and chromosome mapping on the high temperature resistance characteristics of the period, and the number and shape of heat-resistant rice (referred to as QTL) mapped to each chromosome of rice, among which, the most significant genetic effect of a QTL The locus was the line Bala from Pakistan, which explained 18% of the phenotypic variation. However, due to the differences in experimental materials, high-temperature treatment procedures and methods used by different researchers, the experimental data obtained are difficult to confirm and repeat each other, and the QTL intervals located are relatively large, so that it is impossible to determine candidate genes and carry out related research. Molecular cloning and functional complementation verification. Therefore, for the above-mentioned heat-resistant QTL, not only lack of necessary data support in theory, but also difficult to effectively use in production practice. the

In addition, some researchers have also studied rice heat tolerance-related genes by homologous cloning. Yamanouchi et al. (2002) used map-based cloning to locate and clone a rice spot gene Sp17, and found that one of its reading frames was highly similar to heat stress transcription factors. Under heat stress conditions, the expression levels of mutant and wild-type Sp17 Both raised. Yokotani et al. (2008) transferred the rice heat resistance gene encoding OsHsfA2e into Arabidopsis, and the tolerance of the transgenic Arabidopsis to environmental stress was enhanced. Microarray analysis of overexpressed Arabidopsis plants under non-stress conditions found that the expression of some stress-related genes increased, including several types of heat shock proteins. the

It is generally believed that heat shock protein (heat shock protein, HSP) is related to high temperature response. Existing reports have shown that rHsp90 responds to several stresses such as salt, drought, and high temperature, and high temperature treatment at 42°C and 50°C for 30 minutes can significantly increase the expression of rHsp90 (Liu et al., 2006). Another report showed that there are 40 α-crystal-containing genes in rice, of which 23 are heat shock proteins (Sarkar et al., 2009). Microarray and RT-PCR analysis showed that 19 of the 23 heat shock proteins were up-regulated at high temperature. In addition, Chang et al. (2007) transferred rice heat shock protein Hsp101 into tobacco and found that overexpressed plants survived better than wild-type controls at high temperatures. Wu et al. (2009) used the HSP10 promoter to drive the expression of OsWRKY11, and found that the leaves of transgenic rice plants wilted slowly and had a high survival rate after heat treatment. Yokotani et al. (2011) found that transgenic plants were not only resistant to salt stress, but also resistant to drought and high temperature by overexpressing the rice OsCEST (chloroplast protein-enhanced stress tolerance) gene in Arabidopsis. the

Zinc finger protein (Zinc finger protein) is a large family of transcription factors, which are involved in gene expression regulation, cell differentiation, embryonic development and other life processes (Gerisman﹠Pabo, 1997; Laity et al., 2001), especially in stress resistance It plays an important role in the regulation of gene expression (Li ﹠ Chen, 2000). According to the number and position of cysteine (C) and histidine (H) residues in the zinc finger protein, the transcription factors containing the zinc finger protein domain can be divided into C2H2, C2C2, C3H, C3HC4 (that is, RING finger ), C3HC5 (ie LM finger) and other subclasses. Among them, the C2H2 type zinc finger protein is the most clearly studied type of zinc finger protein. It consists of two cysteines and two histidines and Zn2+ to form a coordination bond, and then forms a β sheet and an α helix. Tight finger structure. Kim et al. (2001) isolated the cold-induced zinc finger protein gene SCOF-1 from the soybean cDNA library, and its encoded product contains two typical C2H2 zinc finger structures. The expression of SCOF-1 is specifically induced by low temperature and ABA, while Not induced by salt stress, transgenic studies confirmed that overexpression of SCOF-1 can enhance cold tolerance in Arabidopsis and tobacco. Liu Mengmeng et al. (2007) cloned a soybean C2H2-type zinc finger protein transcription factor gene GmC2H2. The expression of GmC2H2 is related to the stress induction of low temperature and ABA. As for C3HC4-type and CHY-type zinc finger proteins, there are only reports of successful isolation in Arabidopsis, rice, Physcomitrella bulbarum, Artemisia salsa, corn, pineapple, soybean and other plants (Stone et al., 2005; Ohyanagi et al. ., 2006; Rensing et al., 2008; Yang et al., 2008; Alexandrov et al., 2009; Yang Xiangyan et al., 2009; Wu Xuechuang et al., 2010). The AdZFP1 gene of Artemisia annua is a typical example of encoding this type of zinc finger protein (Yang et al. 2008). Semi-quantitative PCR analysis showed that AdZFP1 gene was strongly induced by exogenous ABA, and to some extent induced by high salt, low temperature and high temperature. . Wu Xuechuang et al. (2010) also screened a C3HC4-type zinc finger protein gene GmRZFP1 from the soybean drought cDNA library. The research results showed that this gene is mainly induced by high temperature and drought. When high temperature stress is 1-6 hours, GmRZFP1 The expression level of the gene was positively correlated with the treatment time, and it decreased after 12 hours of stress, and the expression level was the highest at 24 hours. These findings thus suggest that the GmRZFP1 gene is induced by multiple stress treatments and may be involved in multiple stress signaling. the

In addition, Huang et al. (2008) found 12 A20/AN1 type zinc finger proteins in japonica rice. Microarray analysis found that low temperature, drought and H2O2 respectively induced four genes (ZFP177, ZEP181, ZFP176, ZFP173), two genes (ZEP181 and ZFP176) and one gene (ZFP157) was expressed. Further studies showed that ZFP177 responded to both low temperature and high temperature stress. Overexpressing the ZFP177 gene, the transgenic tobacco obtained can tolerate a low temperature of 2°C and a high temperature of 55°C, but is more sensitive to salt stress and drought stress, which shows that ZFP177 plays an important role in various abiotic stresses in plants , but different stresses may have different response mechanisms. the

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Summary of the invention

In order to solve the above problems, the inventors of the present application have developed an optimized method for positioning plant stress resistance genes, especially high temperature resistance genes, through dedicated research, providing a variety of new gene screening, positioning markers, and precise positioning A new high temperature resistance gene of rice has been discovered, thereby providing a breeding method for restoring temperature sensitive plants to non-temperature sensitive plants and transforming ordinary plants into high temperature resistant plants. Specifically, the present invention relates to the following aspects:

1). A polypeptide, which is selected from any of the following:

A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO.3;

B) The amino acid sequence contained in the amino acid sequence shown in SEQ ID NO.3 or obtained by deletion, substitution, or insertion of one or more amino acids, and the polypeptide has the function of anti-high temperature;

C) a polypeptide having the amino acid sequence shown in SEQ ID NO.3;

D) the amino acid sequence of the polypeptide shown in SEQ ID NO:3.

2). A gene The polynucleotide sequence of the gene is a polynucleotide sequence selected from any of the following:

A) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO.1;

B) Contains the nucleotides corresponding to positions -1395 to -1406 in the nucleotide sequence shown in SEQ ID NO.1 as GGGGGGGGGGGG remains unchanged, and is deleted in other nucleotide sequences, The polynucleotide sequence obtained by replacing or inserting one or more nucleotides, and the encoded polypeptide has the function of anti-high temperature;

C) a polynucleotide having the nucleotide sequence shown in SEQ ID NO.1;

D) nucleotide sequence such as the polynucleotide shown in SEQ ID NO:1;

E) A polynucleotide sequence encoding the polypeptide of any one of claim 1.

3). A vector comprising the gene of item 2. the

4). A host cell, characterized in that the cell comprises the polypeptide described in item 1, or the gene described in item 2, or the vector described in item 3, the host cell is preferably a eukaryotic cell, and more Preference is given to plant cells and yeast cells, preferably grass cells, particularly rice ( Oryza sativa L.) cells.

5). Molecular markers for screening, locating and isolating new nucleotide sequences, the markers are selected from:

A) Insertion/deletion marker InDel5, which is located between 9130-9150kb from the short arm end of rice chromosome 9, and the amplified product of the marker has a length polymorphism;

B) SNP marker, referred to as RBsp1407, in high temperature sensitive rice and high temperature resistant rice plants, based on the polynucleotide sequence described in SEQ ID NO: 1, the corresponding sequences of the markers are TGT-3060ACA and TGG-3060ACA, respectively;

C) Microsatellite DNA marker RM7364, which is located between 9440-9450kb from the short arm of rice chromosome 9, and the amplified product of the marker through primers has length polymorphism.

6). The application of the marker described in item 5 in screening, locating, and isolating high temperature resistance/sensitivity genes, wherein the high temperature is preferably 42°C or above, 45°C or above, 48°C or above, or 50°C or above . the

7). The plant breeding method, the method comprising the gene described in item 2, or the polypeptide described in item 1, or the vector described in item 3, or the host cell described in item 4, or item 5 or 6 said markup. the

8). A method for restoring temperature-sensitive rice to non-temperature-sensitive rice, the method comprising genetically manipulating the nucleotide corresponding to position -3060 of SEQ ID NO: 1 in chromosome 9 of the temperature-sensitive rice genome Mutate to G. the

9). A method for inducing a high temperature resistance phenotype in rice, the method comprising inserting G into the 5′ end corresponding to position -1394 of SEQ ID NO: 1 in chromosome 9 of the rice genome that does not have a high temperature resistance phenotype through genetic manipulation , wherein the high temperature is preferably 42°C or above, 45°C or above, 48°C or above, or 50°C or above. the

10). A method for making temperature-sensitive rice a high-temperature-resistant rice, the method comprising mutating the nucleotide corresponding to position -3060 of SEQ ID NO: 1 in chromosome 9 of the temperature-sensitive rice genome through genetic manipulation to G, and a G is inserted upstream of the 5' end of position -1394 in the temperature-sensitive rice genome chromosome 9, so that the resulting nucleotide sequence is the same as that of the nucleotide sequence shown in SEQ ID NO.1 Nucleotides from -1395 to -1406 are the same, that is, the sequence away from the start codon from -1395 contains 12 consecutive Gs, wherein the high temperature is preferably 42°C or above, 45°C or above, and 48°C or above, or 50°C or above. the

11) The present invention relates to a method for identifying the high temperature resistance characteristics of plants. The method can be summarized as follows: that is, the seedlings are cultivated in the soil at the stage of two leaves and one heart to three leaves and one heart stage, and are subjected to high temperature treatment at 45-48°C for 79 hours, and after recovery for 5 days Observe the temperature response results. As a standard procedure for high temperature identification, relative humidity and other plant conditions are set at the same level. the

12) The method described in item 11 above preferably also includes the pots used each time are 43cm long, 33cm wide and 10cm high, and the potting soil is weighed and added in equal amounts. The cultivation specification is 5 rows per variety, and each 12 plants were planted in a row, and 1 row of high-temperature material was planted as a protection row to eliminate the marginal influence, and a clear boundary was reserved between the protection row and the formal experimental materials; the humidity in the growth chamber was set at 75% during treatment, and the light-dark cycle rotation The duration is set to 12h. the

Description of drawings

Fig. 1 High temperature stress treatment test and the high temperature resistance response of resistant and susceptible varieties HT54 and HT13. The figure shows the growth status of soil-cultured seedlings at the two-leaf and one-heart stage after 79 hours of different high-temperature treatments and recovery for 5 days. A: 42°C high-temperature treatment; B: 45°C high-temperature treatment; C: 48°C high-temperature treatment; From the photos in the figure, it can be seen that the heat shock response of the high temperature resistant variety HT54 and the high temperature sensitive variety HT13 to the high temperature treatment of 45°C and 48°C showed qualitative differences in survival and death. the

Figure 2 is the linkage genetic map of microsatellite molecular markers (SSLP) established by mapping high temperature resistance genes in rice. The distance between the markers is the physical distance. the

Fig. 3 Preliminary linkage group detection results of high temperature resistant loci in the F2 generation of HT54 (anti)X HT13 (sensible) hybrids. The molecular marker used was the SSR marker RM444 located in the ninth linkage group of rice. In the figure, M represents the molecular weight marker; RP is the high-temperature-resistant parent; SP is the high-temperature-sensitive parent; RB is the extreme high-temperature-resistant single-plant DNA pool; SB is the high-temperature-sensitive extreme single-plant DNA pool; F2 recessive high temperature extreme single plant. the

Fig. 4 Linkage genetic map of high temperature resistance gene OsHTAS on chromosome 9. (a): Linkage genetic map generated from 61 mapped populations; (b): Linked genetic map generated from fine mapping of 131 mapped populations to the black segment in (a). the

Figure 5. Using a single nucleotide polymorphism (SNP) conversion in the candidate gene (ZFP) sequence between the high-temperature-resistant and high-temperature-sensitive parents, the PCR-RFLP markers appear in the mapping population when the InDel5 and RM7364 markers are used for mapping The results of the detection of the exchanged strains. In the figure, M, RP, SP, S represent the molecular weight marker (DL2000), the high temperature resistant parent HT54, the high temperature sensitive parent HT13 and the three exchanged strains, respectively. the

Fig. 6 Dynamic expression pattern analysis of ZFP resistant and susceptible alleles to 45°C high temperature stress treatment. The analysis method used is RT-PCR semi-quantitative analysis. The number of PCR amplifications was 25 cycles, and the rice actin gene actin was used as an internal standard. In the figure, the four rows from top to bottom are HT54 (anti)/actin, HT13 (sense)/actin, HT54 (anti)/ZFP and HT13 (sense)/ZFP. As can be seen in the figure, ZFP was up-regulated and down-regulated in the high temperature-resistant and sensitive samples treated for 6 hours, respectively. the

Fig. 7 Subcellular localization analysis of the high temperature resistant gene OsZFP coding sequence. The figure shows that compared with the control 35S-YFP, 35S-ZFP-YFP, the fusion protein of the high temperature resistance gene coding product and yellow fluorescent protein, is mainly located on the cell membrane.

Fig. 8 The high temperature resistance gene OsZFP DNA sequence, coding sequence and its product amino acid sequence involved in this application.

Detailed description of the invention

In order to solve the above-mentioned problems, the present inventors have completed the present invention on the basis of a large number of test results after earnest research. The present invention will be described in detail below in conjunction with the accompanying drawings.

1. Standardized identification procedure for rice seedling high temperature tolerance

First, the inventor established a standardized identification procedure for high temperature tolerance of rice seedlings. The outstanding advantages of this identification program are clear classification of resistance and sensitivity, good reproducibility of results, and strong practical applicability. Therefore, the establishment of this program will provide a good technical basis for the screening and evaluation of rice high-temperature-resistant germplasm resources, genetic analysis of high-temperature-resistant genes, chromosome mapping and cloning. Please refer to the content described in Example 1 for specific identification content.

2. Molecular mapping of rice high temperature resistance genes

On the basis of the above test results, the inventors used the high-temperature-resistant rice material HT54, the high-temperature-sensitive rice material HT13 and their hybrid offspring F1 and F2 as exemplary test materials to carry out molecular mapping of rice high-temperature resistance genes.

experiment method

(1). Screening of parental polymorphic markers

The molecular mapping of high temperature resistance genes in rice was carried out using microsatellite DNA length polymorphism molecular markers (SSLP) with high polymorphism and good repeatability. The SSR primers used were based on the Gramene database (www.gramene.org) and NCBI (www. ncbi.nlm.nih.gov) database and synthesized by Shanghai Bioengineering Technology Service Company.

Rice genomic DNA was extracted using a small amount of DNA extraction method reported by McCouch et al. (1988). The specific method steps are as follows: 1) Cut a small piece of leaf 4-5 cm, add 700μL 1.5 × CTAB (containing 1.5% CTAB, 75 mM Tris-HCl, 15mM EDTA, 1.05 M NaCl), fully grind; 2) Grind the Transfer the homogenate to a 1.5 ml centrifuge tube, cool to room temperature in a water bath at 56°C for 20 min; 3) Add an equal volume of chloroform:isoamyl alcohol (24:1) and shake well; 4) Centrifuge at the highest speed (13200 rpm) for 10 min; 5) Transfer the supernatant to a new centrifuge tube, add twice the volume of pre-cooled 100% ethanol, and centrifuge to collect DNA after standing still for 20 min; 6) Remove the supernatant, air-dry the DNA, and add 50-100 μL Double-distilled water, tested in the ultraviolet spectrophotometer, dilute the DNA concentration, and prepare a set of DNA working solution, the concentration of which is about 50-100ng/μL, and store it in the refrigerator at 4°C for later use. the

2) Construction of targeting groups

In the autumn of 2008, a hybrid combination was prepared in the experimental base of the Rice Institute of Guangdong Academy of Agricultural Sciences, using the high temperature resistant material HT54 as the male parent and the high temperature sensitive material HT13 as the female parent. The female parent HT13 was treated with the method of "killing males in warm soup". Considering that HT13 is a material sensitive to temperature, the temperature for killing males in warm soup was set 2°C lower than usual to avoid damage to its pistils. The specific method is: at the earing stage of rice, at around 7:30 in the morning on a sunny day, select the ears of the female parent HT13 (take out 2/3), turn the thermos bottle with the water temperature adjusted to 43°C upside down to cover the ears, and plug them with cotton Keep warm at the mouth of the bottle. After 8 minutes, take out the tassels and shake off the water drops. If some spikelets (have bloomed and pollinated or will bloom after a few days) will not open, they should be cut off and put in a paper bag immediately. Take the male parent around 9:00 For the tassels of HT54, the stalks were inserted in a bottle filled with tap water and covered with a black cloth. After one hour, most of the florets on the male parent were in full bloom, and the pollen was shaken off on the surface that had been killed by high temperature and had good glumes. Repeat pollination 2-3 times on the stigma of the ear. Immediately after pollination, put a kraft paper bag on the mother ear, and clamp the bag mouth with a paper clip, but do not clamp the ear stem, hang a sign, record the name and hybridization date, and take off the paper bag after about 8 days, and harvest F1 seeds when they are mature .

In the winter of 2008, the hybrid F1 was planted in Hainan. After the plants grew up, the leaves were taken to extract DNA, and the polymorphic SSR marker 3-4 pair of the two parents was used to test whether the plants were true hybrids, and the plants were bagged and self-harvested after heading. F2 seeds were used for subsequent identification of high temperature resistance, and the genetic analysis of high temperature resistance genes was carried out through the resistance and sensitivity ratios after high temperature treatment, and the high temperature resistance genes were mapped. the

3) High temperature treatment procedure

The parents, F1 and F2 were subjected to high temperature treatment according to the aforementioned seedling culture method and standardized high temperature treatment procedure. The specific steps are: a. Prepare seedling pots, that is, add an equal amount of paddy field soil that has been sieved and fully mixed into each pot of seedling pots (43cm×33cm×10cm in size); b. Sowing, that is Sow the germinated seeds on demand in pots, 11 rows/pot, 18 seeds/row. At the same time, sow 1 row/dish of each parent as a control, and set up 2 rows of protection rows around it. Fertilizer and water management is the same as general potted rice management; C. Sampling. When the seedlings grow to the three-leaf stage, take the second leaf about 2-3cm from each plant and place it in a -80°C refrigerator for DNA extraction from sensitive individual plants after identification. D. After the seedlings resume growth for 2 days, that is, the day before the third leaf is about to grow completely, move them into an artificial climate box for high temperature treatment. The settings of the artificial climate chamber are as follows: 33°C 1h (Light, abbreviated as L, the same below) → 36°C 1h (L) → 39°C 1h (L) → 42°C 1h (L) → 45°C 1h (L) → 48°C 7h (L) → 48°C 12h (Dark, abbreviated as D, the same below) → 48°C 12h (L) → 48°C 12h (D) → 48°C 12h (L) → 48°C 12h (D) → 48°C 12h (L). Humidity 75%.

(4). PCR reaction procedure and detection

The SSR analysis was mainly revised based on the method of Wu et al. (1993). The PCR system was 25 μL: 2.5 μL 10× buffer (Mg2+); 0.5 μl 10Mm dNTP; 1.0 μl 5 μM 5′-primer; 1.0 μl 5 μM 3′- Primers; 0.5 μl Taq polymerase; 1.0 μl DNA; 18.5 μl ddH ₂ O. dNTP was ordered from Shanghai Sangong, and Taq polymerase was ordered from Dingguo. The PCR amplification program is: 94°C pre-denaturation for 5 minutes, then start 35 cycles (94°C denaturation for 45s, select a suitable temperature for primers to anneal at 50-60°C for 45s, 72°C for 45s extension), and then continue to extend at 72°C for 10 minutes . Amplified products were detected by 3% agarose gel electrophoresis (AGE) or 4% denaturing polyacrylamide gel electrophoresis (PAGE).

(5). Preliminary mapping of high temperature resistance genes

The F2 generation segregation population was selected to construct the mapping population, and the recessive extreme population method was used for gene positioning, and the molecular marker used was microsatellite DNA polymorphism molecular marker (SSLP). The specific procedure is as follows: firstly, the F2 segregation population is subjected to high-temperature treatment, and individual plants sensitive to high temperature are screened out to be used as a mapping population for gene positioning. Then, from the F2 segregation population, 10 high-temperature-resistant and high-temperature-sensitive plants were randomly selected, and the leaves were mixed in equal amounts and ground to extract DNA, which was used to construct sensitive and high-temperature-resistant DNA pools. After that, using the polymorphic SSLP markers between the parents (Chen et al., 1997; Temnykh et al., 2000), the two DNA pools were first analyzed for markers, and the polymorphisms in the two DNA pools were screened out. Markers, whereby the linkage group where the gene is located is preliminarily determined. On this basis, the polymorphic markers obtained in the DNA pool were used to perform genotype determination on the mapping population constructed by the recessive extreme individuals in the above-mentioned F2 segregation generation one by one, and the obtained data were used for the preliminary positioning of the target gene.

(6). Fine mapping of high temperature resistance genes

On the basis of the preliminary positioning, the marker encryption analysis is further performed on the positioning segment to make the length of the positioning segment at least. The SSLP marker used can be obtained from the Poaceae Genome Database (http://www.gramene.org), Or design and synthesize it yourself based on the genome sequence until such markers are used up on the linkage groups involved in the target gene. Then, biological function prediction and molecular characterization (including motifs, functional domains, conserved sequences, and regulatory sequences, etc.) analysis of the coding sequence in the marker interval (generally at least 0.5Mb) is performed using biological information, so that Candidate genes were identified. Afterwards, specific primers were designed for the candidate genes, and the candidate genes were cloned and sequenced by TA cloning, and the target genes were predicted according to whether there were differences.

(7). Calculation of genetic distance

The results of the electrophoresis pictures were converted into numerical statistics: the ones consistent with the band type of HT54 were marked as A, those consistent with the band type of HT13 were marked as B, those with both parental bands were marked as H, and those without bands were marked as -.

Linkage genetic analysis was carried out using MAPMAKER3.0 software (Lander et al, 1987) to calculate the genetic distance (cM), and genetic mapping Mapdraw2.1 (Liu Renhu and Meng Jinling, 2003) was used to locate and analyze the anti-high temperature genes. the

In order to precisely locate the gene, the inventor has also developed a new localization marker. For the preparation example of the marker, please refer to Example 2 below. the

Genetic analysis and gene mapping results

1) Parental polymorphism screening and molecular map establishment

A total of 2304 SSLP molecular markers were used to screen the parents for polymorphism, and 322 SSLP marker molecules with polymorphism among the parents were screened out. Their distribution on the chromosome is measured by physical distance as shown in Figure 6, except for the first Except for chromosome 9 and near the centromere, the obtained polymorphic SSLP markers covered each chromosome more evenly.

The linkage genetic map of microsatellite molecular markers (SSLP) established by mapping high temperature resistance genes in rice is shown in Figure 2. The distance between the markers is the physical distance. the

2) Genetic analysis confirmed that the high temperature resistance of HT54 at the seedling stage is controlled by a dominant single gene

The parents, hybrids F1 and F2 were subjected to high temperature treatment at the two-leaf one-heart stage using a standardized high-temperature treatment program. The results showed that the F1 plants survived the high temperature treatment completely, and the F2 population showed obvious separation of resistance and sensitivity. Among them, there were 442 high temperature resistant plants and 152 high temperature sensitive plants, and the resistance and sensitivity separation met the segregation ratio of 3:1 (see Table 1), chi-square test, P>0.05, indicating that the difference did not reach a significant level. This result therefore shows that the high temperature resistance of HT54 at the seedling stage is controlled by a dominant single gene, which is now named OsHTAS ( O ryza s ativa Heat Tolerance a t Seedling stage).

Table 1 Results of genetic analysis of HT54 high temperature resistance characteristics

3) Preliminary mapping shows that the high temperature resistance gene is located in rice linkage group 9

Among the 322 SSLP markers with polymorphism between parents, it was found that only RM444 showed a heterozygous band pattern when detecting the high temperature resistant DNA pool, and the high temperature sensitive DNA pool was consistent with the sensitive parent, which indicated that RM444 may be linked to the target gene (see Figure 3). In addition, in the PCR amplification product of F1, the amplification intensity of the two parental bands is basically the same; the band type of the high temperature resistant DNA pool is heterozygous, but the band type of HT54 is obviously stronger than that of HT13, implying that the band type of high temperature resistant DNA The pool may contain two types of high temperature resistant homozygous and heterozygous; while the band type of the high temperature sensitive DNA pool is basically HT13 band type, indicating that the DNA pool is mainly composed of DNA of recessive homozygous individual plants sensitive to high temperature, thus indicating that the used Molecular markers may be linked with target high temperature resistance genes. Therefore, 61 recessive extreme high-temperature-sensitive individuals and 40 high-temperature-resistant individuals were selected from the high-temperature-treated F2 population to construct two verification populations for RM444 marker analysis. The results showed that among the 61 invisible extreme high temperature individual plants, 13 were of the heterozygous band type, 43 were of the HT13 band type, 5 were of no band type, and 0 were of the HT54 band type; and 23 of the 40 high temperature resistant plants It is heterozygous banding type, 2 strains are HT13 banding type, and 15 strains are HT54 banding type. These results thus further verified that the RM444 marker is linked to the high temperature resistance gene OsHTAS.

4) Fine mapping further locates the high temperature resistance gene between two molecular markers RM7364 and InDel5, the actual physical distance of this interval is 420bp

From the database on the website www.gramene.org, search for the primers at the short arm end of chromosome 9 (that is, above RM444) and analyze the parental markers. A total of 3 SSLP markers with polymorphisms between the parents were found. They were respectively It is RM23687, RM23719 and RGNMS2991. After using these three markers to scan the previously used 61-strain mapping population, it was found that RM23719 and RM23687 were indeed linked to the high temperature resistant OsHTAS gene (Fig. In addition, it was also found that RM23719 is located immediately below RM444, the genetic distance between the two is 0.9 cM (Figure 4a), but the actual physical distance between the two is 4.48Mb; while RM23687 is close to the endpoint (the distance between the two is 1.07Mp), It is 1.01Mb away from RM23719, but the detected linkage genetic distance with the high temperature resistance gene OsHTAS reaches as much as 17.9 cM. According to the information on rice chromosome 9 listed in the Gramineae Genome Database, it was found that the above abnormal linkage inheritance may be due to the proximity of RM23719 to the centromere. Therefore, it is speculated that the high temperature resistance gene may be below RM444, that is, the long arm of rice chromosome 9.

Next, search for primers at the long arm end of rice chromosome 9 (below RM444) from the Poaceae genome database, and find 114 pairs of SSLP primers. After parental polymorphism analysis, a total of 10 pairs of primers with parental polymorphisms were found. They are RM23982, RM23985, RM7364, RM24019, RM5777, RM240712, RM24075, RM24099, RM24102, and RM24170. The results of marker analysis of the resistant and sensitive DNA pools showed that: RM23982, RM23985, RM7364 and RM24019 also showed polymorphisms between the resistant and sensitive DNA pools, and then these markers were used for further marker analysis of the 61 strains of the F2 positioning population, and then The high temperature resistance gene OsHTAS was positioned between RM23985 and RM7364 (see Figure 8b). In the absence of further known SSLP markers in this interval, 90 pairs of insertion/deletion (Insertion/Deletion, InDel) markers were designed based on the sequence information of the indica and japonica rice genome. After marker analysis of the parents, a total of Eight pairs of primers with parental polymorphisms were found, but only InDel3 and InDel5 had large differences between the parents and were easy to identify the band patterns of the mapping population. the

The linkage genetic map of the high temperature resistance gene OsHTAS on chromosome 9 is shown in Figure 4. the

The primer sequences for these two insertion/deletion markers are, InDel3F: 5′-GTTTGCG ACATTGGAGCCTTC-3′ and InDel3R: 5′-AATGCTTGGGTATGCTAG GTGAA-3′; InDel5F: 5′-TCCTCGGAGATGTTTGACCTTG-3′ and InDel5R: 5′- CAGAAGGTG TACGCAACTCTTGT-3′. the

Therefore, using these two self-designed indel markers, the rice high temperature resistance gene OsHTAS was further located between RM7364 and InDel5, and the genetic distance between the gene and these two closely linked markers was 2.5cM and 1.7cM, respectively. The actual physical distance between them is 420Kb. Then, the mapping population was expanded to 131 strains, and the genetic linkage distances detected between InDel3, InDel5, RM7364, RM24019 and the high temperature resistance gene OsHTAS were 4.0 cM, 3.2 cM, 1.2 cM and 1.6 cM, respectively (Fig. 4b). These mapping results are basically consistent with those of the aforementioned 61 strains mapping populations. the

5) The identified candidate gene is the zinc finger protein gene, which has two SNP differences between the heat-resistant and sensitive parents, and co-segregates with the PCR-RFLP marker developed by one of them

Since it is difficult to obtain available polymorphic markers in the above positioning interval, the existing biological database information combined with published genetic data is used to screen and determine candidate genes. From the http://rice.plantbiology.msu.edu/ website, search the DNA sequence of the 420Kb long positioning segment (from Indel5 to RM7364) to find a total of 60 known and unknown genes, of which 15 are coding reverse transcription Among the genes of transposon proteins, 6 are genes encoding transposon proteins, 27 are genes encoding known functional proteins, and 12 are genes encoding unknown functional proteins. The results of the existing reports showed that the related ubiquitin-conjugating enzyme protein (LOC_Os09g15320) and zinc finger protein (LOC_Os09g15430) genes were related to high temperature tolerance in rice, therefore, they were initially identified as candidate genes; then, we normalized The microarray data were analyzed, and the genes expressed more than 100 in 8 seedling stages [nucleobase-ascorbic acid transporter (LOC_Os09g15170), retrotransposon protein (LOC_Os09g15250), ubiquitin-binding protein (LOC_Os09g15320), transporter family protein (LOC_Os09g15330) , hydrolase (LOC_Os09g15340), NAD-dependent epimerase/dehydratase (LOC_Os09g15420), zinc finger protein (LOC_Os09g15430), and the serine/threonine-rich protein T10 in the DGCR region (LOC_Os09g15480)], the ubiquitin-binding protein (LOC_Os09g1532) and zinc finger protein (LOC_Os09g15430) are highly expressed, indicating that these two genes play an important role in the growth and development of rice seedlings. Therefore, the cDNA sequences of these two candidate genes and the genomic DNA sequences of the promoter and terminator were amplified from the seedling leaf tissues of the resistant and susceptible parents for sequencing. It was found that the cDNA sequences of the resistant and susceptible parents were not different, but the zinc finger There is a single base polymorphism (SNP) difference in the 5' untranslated region of the protein (LOC_Os09g15430) and its promoter sequence. Due to the SNP difference in the promoter sequence, a restriction endonuclease Bsp1407 recognition site (T↓GTACA) was changed, so a PCR-RFLP (CAPs) marker was designed, denoted as RBsp1407.

The size of the PCR amplification product of the marker is 580bp. The amplified PCR product was recovered, and then the fragment was digested with endonuclease Bsp1407Ⅰ (Promega). The results showed that the amplified product of HT54 could not be digested, and the fragment size was still 580bp, while HT13 and 131 All the exchanged strains presented in the strain mapping population could be digested, and two fragments of 422bp and 158bp were produced after cutting (Figure 5). The test results of the PRBsp1407I marker showing zero crossover strains between InDel5 and RM7364 indicated that the marker and the high temperature resistance gene OsHTAS were co-segregated under the conditions of the used mapping population size, thereby further identifying the candidate gene as a zinc finger protein gene. the

The technical solutions of the present invention are described in detail below by means of specific examples. Those skilled in the art understand that the specific implementation is to facilitate those skilled in the art to reproduce the exemplary technical information provided by the present invention, which is not a limitation to the claims of the application for protection, based on the exemplary test plan As long as the technical solutions and the beneficial effects achieved do not deviate from the gist of the present invention, the changed technical solutions shall be included in the protection scope of the present application. the

Example

Example 1 Identification of High Temperature Resistance Genes

In this example, high temperature tolerant rice variety HT54 and high temperature sensitive rice variety HT13 (both of which are O. sativa ssp . indica) were used as exemplary test materials.

1) Cultivation of rice seedlings and setting of high temperature resistance treatment conditions

(1). Seedling cultivation

Take the paddy field soil from the same plot, air-dry it naturally, crush and sieve it, divide it into plant growth pots (26cm×18cm×6cm) according to the same weight, add the same amount of water to soak overnight, and then use it for sowing. The test materials were sown after soaking and germination. Each pot is divided into two parts, one half is planted with HT54, the other half is planted with HT13, each variety has 3 rows, and each row has 8 plants.

(2). High temperature treatment period, treatment temperature and other conditions settings

The high temperature treatment period is the seedling stage. The artificial climate box used is the intelligent artificial climate box PRX-1000B produced by Zhejiang Ningbo Fu Experimental Instrument Factory. The temperature setting is divided into three treatments: 42°C, 45°C and 48°C. Start to rise at a rate of 3°C/h until it reaches the set high temperature, the humidity used is 75%, and other cultivation conditions are set at the same level.

(3). Identification procedure and standardized parameter setting of rice high temperature resistance

The rice seedlings of rice varieties HT54 and HT13, which are resistant to high temperature and sensitive to high temperature, were grown by soil culture to the two-leaf-one-center and three-leaf-one-center stages, and moved into the growth box, and subjected to high-temperature treatment at three temperatures for 79 hours. After the treatment was completed and recovered for 5 days, the seedlings were observed response to high temperature. The result is shown in Figure 1. It can be clearly seen from the results in Figure 1 that: when the temperature was 42°C, the treated high-temperature-resistant and sensitive seedlings survived completely; and when the treatment temperature was 45°C and 48°C, the treated high-temperature-resistant and sensitive Varieties showed qualitative differences in survival and death (Figure 1), but obviously 45°C is not the upper limit temperature that the high-temperature-resistant variety HT54 can tolerate, so 48°C was finally selected as the temperature setting value of the standard program. Therefore, the high temperature resistance identification procedure of rice seedlings determined by this experiment can be summarized as follows: soil-cultured seedlings with two leaves and one center, treated at 48°C and 75% relative humidity for 79 hours, and after 5 days of recovery, observe the response of the seedlings to the high temperature. And the survival is used as the identification index of rice resistance and sensitivity to high temperature. From the photos in the figure, it can be seen that the heat shock response of the high temperature resistant variety HT54 and the high temperature sensitive variety HT13 to the high temperature treatment of 45°C and 48°C showed qualitative differences in survival and death.

Example 2 Confirmation of new gene markers

In order to realize the precise positioning of high temperature resistance genes in rice, it is obvious that the SSLP in the prior art is not enough to meet the needs. To this end, the inventors have developed new positioning markers. Specifically, the inventors searched for insertion/deletion polymorphisms (Insertion/Deletion, InDel) through differences in the whole genome sequences of the japonica rice Nipponbare and the indica rice 9311 published by NCBI (http://www.ncbi.nlm.nih.gov/) location. According to the DNA sequence at both ends of this site, use Primer5.0 to design appropriate primers, and use NCBI online Primer blast to ensure the specificity of the primers.

TA cloning and sequencing analysis

a) Adding A reaction: Since the purified PCR product is amplified with Proybest enzyme, its end is blunt, and base A needs to be added for TA cloning. The total system for adding A reaction is 20 μL, and the components and volumes to be added are: 10×PCR buffer, 2 μL; Taq enzyme, 0.5 μL; dNTPs (10 mM), 0.5 μL; PCR purified product, 17 μL. Mix well and put it on the PCR instrument, 72°C for 30min, and store at 4°C. Add A reaction products were purified using the Axy Prep PCR Clean-up Kit (Axygen).

b) Connection: operate according to the instructions of the pMD18-T vector system kit (TaKaRa). Take 4 μL of the purified reaction product of adding A, mix with 5 μL of the ligation mixture (solution I) and 1 μL of the pMD18-T carrier, connect at 16°C, and keep the temperature for 1-2 hours (performed in a thermostat). the

C) Escherichia coli heat shock transformation of the ligation product: Take competent cells from -80°C, add 10 μL ligation reaction solution, shake gently, and place on ice for 30 minutes; heat shock in a water bath at 42°C for 90 seconds, and immediately place on ice for 2 minutes; Afterwards, add 800 μL of LB medium to each tube, and incubate with low-speed shaking on a shaker at 37°C for 45 minutes (to revive the bacteria, the rotation speed should not exceed 190rpm); after enriching the above bacterial solution, spread it on the LB screening plate (Amp/IPTG/X-Gal ); after the bacterial solution was completely absorbed by the culture medium, invert the culture dish and incubate overnight at 37°C. the

d) Identification of positive clones of transformants: pick white colonies, and carry out PCR amplification identification using vector sequencing universal primers. The PCR reaction system is the same as above, and the reaction program is: 94°C for 5 minutes for pre-denaturation, followed by 95°C for 30 s, 55°C for 30 s, and 72°C extension (the specific time is determined according to the size of the target gene), a total of 30 cycles, followed by 72°C extension for 5 minutes. the

e) PCR product electrophoresis identification: the length of the PCR product of the negative colony is 156bp (only the sequence amplified on the vector), and the fragment of the PCR product of the positive colony is greater than 156bp (the length of the target gene fragment + 156bp). the

F) Sequencing of positive clones: Take 5 mL of LB liquid medium containing 50 μg/mL Amp into a 50 mL centrifuge tube, inoculate colonies with positive PCR results, and culture overnight at 37°C with shaking at 220 rpm; In the tube, centrifuge at 12000rpm for 2min, remove the supernatant, and place the centrifuge tube upside down on absorbent paper to make the bacterial pellet as dry as possible; use the Axygen plasmid extraction kit to extract plasmid DNA. The extracted plasmid DNA was stored at -20°C for later use, and at the same time, 10 μL of the bacterial liquid corresponding to the plasmid was taken for sequencing verification (Shanghai Yingjun). Three positive transformants were verified by sequencing of each gene. The new marker of the present invention is developed from a SNP present on the promoter of the candidate gene in the high temperature-resistant and sensitive parents. Specifically, the appearance of this SNP led to a change in the recognition site of a restriction endonuclease Bsp1407 I, whose sequence on the high temperature resistant parent was 5'-TGGACA-3', which could not be recognized by Bsp1407 I, The sequence on the high-temperature parent is 5'-TGTACA-3', which can be recognized by Bsp1407 I; therefore, specific primers are designed for the region where the SNP is located, the corresponding genomic fragment is amplified, and then digested with Bsp1407 I to detect this polymorphic differences.

According to this principle, the specific primer sequences designed in this example are, BspF: 5'-CCATCCAAACACGCCCTAA-3' and BspR: 5'-ATTGCCCCTTGCTATGG T-3', the size of the PCR-amplified product is 580bp, which comes from the PCR of the high-temperature sensitive parent The sizes of the two fragments obtained after the product was digested by Bsp1407 I were 422bp and 158bp respectively, and the PCR-RFLP marker developed therefrom was marked as RBsp1407.

This marker has been confirmed to co-segregate with the target gene during the localization of high temperature resistance genes. the

In the test of confirming the RBsp1407 marker, the inventors used this marker to perform confirmatory detection on the three recombinant individual plants that appeared after the analysis of the two closely linked markers RM7364 and InDel5 in the recessive extreme positioning population. The results of parental agreement are shown in Figure 5. Thus, the inventors have also developed an insertion/deletion marker InDel5, which is located between 9130-9150kb from the short arm end of rice chromosome 9, and the marker has a length polymorphism between the resistant and susceptible parents; and microsatellite DNA The marker RM7364 is located between 9440-9450kb from the end of the short arm of rice chromosome 9. For the marker RM7364, primers RM7364F: 5'-TTCGTGGATGGAGGGAGTAC-3' were designed; and RM7364R: 5'-RGCGTTTGTAGGAGTGCCAC-3', similarly, it was found that the amplified products of the markers between the resistant and sensitive parents had length polymorphism . the

Example 3 Confirmation of high temperature resistance gene OsHTAS

Through the aforementioned gene mapping research, the inventors discovered a new gene located on chromosome 9 of the rice genome, namely the dominant high temperature resistant gene OsHTAS . The difference in the DNA sequence between the dominant high temperature resistance gene OsHTAS and the recessive allele Oshtas is only a single nucleotide polymorphism (SNP), which occurs in the 5'-end untranslated region of OsHTAS and the high temperature resistance , salt tolerance and drought tolerance-related motifs (11T12G), OsHTAS (TTTTTTTTTTTTGGGGGGGGGGGG) has one more G than Oshtas (TTTTTTTTTTTTGGGGGGGGGGG); dominant high temperature tolerance gene (LOC_Os09g15430) OsHTAS genome sequence full length 4784bp, coding sequence (CDS) full length 1245bp, the coding product is 414aa, belongs to zinc finger family cluster protein; rice ( Oryza sativa ) ZFP gene, the specific sequence is shown in SEQ ID NO: 1 in the sequence listing.

The inventors performed insertions, deletions, and substitutions on some sites of the gene during the specific test process, and performed functional verification on the obtained mutants. The obtained mutant has maintained the same anti-high temperature function as the gene shown in SEQ ID NO:1. More specifically, the -1384th to -1395th nucleotides corresponding to the nucleotide sequence shown in SEQ ID NO.1 are GGGGGGGGGGGG and remain unchanged. The polypeptide encoded by the polynucleotide sequence obtained by deleting, replacing or inserting one or more nucleotides still has the function of anti-high temperature. The insertion, deletion, and replacement are carried out according to genetic manipulation methods known in the art, and the number of changed nucleotides is preferably 1-100, more preferably 1-50, 1-20, and more preferably 1-10 nucleosides Sour change. the

In addition, the inventors connected the high temperature resistance gene of the present invention with an inducible promoter, constructed an expression vector, and transformed it into host cells such as yeast DY1455 and Arabidopsis thaliana Columbia , and successfully obtained positive transformants.

Example 4 Response characteristics of high temperature resistance gene OsHTAS to high temperature stress

The high temperature of 45 ℃ was used to treat different high temperature resistant and sensitive varieties continuously for 12 hours, and the RNA was extracted at 8 time points (0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h), and the candidate gene OsHTAS was tested. RT-PCR semi-quantitative expression analysis. The results showed that the expression change patterns of this gene were significantly different in the heat-resistant and sensitive varieties HT54 and HT13. The expression of the gene was significantly up-regulated on HT54 at 6 hours, but it was down-regulated on HT13. The results are shown in Figure 6 The result in the figure shows that the candidate gene has the characteristics of active response to high temperature stress. It can be seen from the figure that the expression of OsHTASP was up-regulated and down-regulated in the high-temperature-resistant and sensitive samples treated for 6 hours, respectively.

Example 5, Functional verification of candidate genes: overexpression analysis

Clone the CDS of the candidate gene and connect it into the overexpression vector driven by the actin promoter ActinI, and then introduce it into the genome of Nipponbare and the sensitive variety HT13 using the Agrobacterium-mediated method, and obtained 5 positive independent transformations respectively body. Afterwards, the soil-cultured seedlings of these positive independent transformants were subjected to high temperature treatment at 48°C for 79 hours at the two-leaf-one-core to three-leaf-one-core stage using the aforementioned artificial climate chamber, and wild-type plants were used as controls. After the treatment, it was removed from the growth box, and the reaction of the seedlings to the high temperature treatment was observed and recorded after recovering for 5 days under normal temperature conditions. The results showed that after high temperature treatment, all the overexpressed plants returned to normal growth like HT54, indicating that the transformed candidate gene did have the characteristic of high temperature resistance.

Example 6, Functional Verification of Candidate Genes: RNAi Knockout

A specific sequence of the CDS region and UTR region of the candidate gene was connected forward and reverse into the RNA interference vector pTCK303, and introduced into the genome of Nipponbare and the sensitive variety HT54 using the Agrobacterium-mediated method, and 5 and 3 positives were obtained respectively independent transformants. Afterwards, the soil-cultured seedlings of these positive independent transformants were subjected to high temperature treatment at 48°C for 79 hours at the two-leaf-mental-three-leaf-one-heart stage using the aforementioned artificial climate chamber, and the wild-type plants were used as controls. After the treatment, remove the growth box and resume growth for 5 days under normal temperature conditions, then observe and record the response of the seedlings to the high temperature treatment. The results showed that all the overexpressed plants died completely like HT13 after the high temperature treatment, which further indicated that the transformed candidate gene did have the property of high temperature resistance.

Example 7, Subcellular localization analysis of the candidate gene OsHTAS

The results of subcellular localization under the confocal microscope of the onion epidermis bombarded by gene guns showed that, compared with the control 35S-YFP, 35S-ZFP-YFP, the fusion protein of the candidate gene and yellow fluorescent protein, was mainly localized on the cell membrane, the results are shown in Fig. 7, this result shows that the product encoded by the candidate gene ZFP is a membrane protein, which is very consistent with the conclusion that a considerable number of membrane proteins participate in stress signal transduction, and illustrates a correlation between subcellular localization and functional expression . The results shown in Figure 7 show that: compared with the control 35S-YFP, 35S-ZFP-YFP, the fusion protein of the candidate gene and yellow fluorescent protein, is mainly localized on the cell membrane.

In summary, the results of the invention disclosed in this application can enhance the understanding of those skilled in the art on the basic genetic laws of high-temperature resistance traits. It will lay a good theoretical and material foundation for the functional analysis of subsequent candidate genes and the effective use in molecular breeding. the

Sequence listing stand-alone text

SEQ ID NO:1 OsHTAS genome sequence;

SEQ ID NO:2 OsHTAS gene coding sequence;

SEQ ID NO:3 OsHTAS protein polypeptide sequence;

SEQ ID NO:4 artificial primer sequence;

SEQ ID NO:5 artificial primer sequence;

SEQ ID NO:6 artificial primer sequence;

SEQ ID NO:7 artificial primer sequence;

SEQ ID NO:8 artificial primer sequence;

SEQ ID NO:9 artificial primer sequence;

SEQ ID NO:10 artificial primer sequence;

SEQ ID NO: 11 Artificial primer sequence.

Sequence Listing

the

<110> Zhejiang University

  Institute of Rice, Guangdong Academy of Agricultural Sciences

the

<120> A high temperature resistance gene in rice

the

<130> CPCH1263656N

the

<160> 11

the

<170> PatentIn version 3.3

the

<210> 1

<211> 4784

<212> DNA

<213> Rice (Oryza sativa L ssp. indica)

the

<400> 1

gagaagcgcc acaggaaaac gagccgcgtc gctttcgcga gagagggaca cctgctctgc 60

the

ttccgcttcc gcttccccct ctcgagtctc tcgcctctcc cccgtggcca accccacacac 120

the

cgcgggttgg aggagggagga ggaggaggta ggggaaatcc ccgtcggccg tcggctcggc 180

the

gccgaatcga tccggtgagt gagtgattga gttcgtctct gctctctctc ctccttgttc 240

the

aattatcaag ctcttgaatc gagtcctagt agtagtgtgc tagaggtgct ggatgatttg 300

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ggtttttggg gatttttttt tttgggggggg gggggttgtt tgggaactgc tagtgcgttt 360

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gtggtatact ggtatgggag ttcgttgcta atggacgggg tatttgggaa cttttagggt 420

the

tttgtttgga tggatttttg gttgtggttt ttgtcaggaa tgggtgtcgg cgacttggtt 480

the

agctttagct tttgagaatt tttttcgccc ctgttgttct gttcatggtt catctttaga 540

the

ttcagaggag aatccttcta gcgtttttct gagaatgaaa cctttttttt tatgttttta 600

the

tttttacggg gctataattt ttccactgcc ttttgtgatt actattacat aattacatgt 660

the

ccctttgatg aataatggat gttggttctt tggctaaaag tcaaattgcg acttgaagta 720

the

gaaacgggtg tacctgggat taagttgcca tcgctggaac tagagttaat tattggtttg 780

the

atcattgttg cttcgcgttg gatatactat cggtttagca gttagcatga tactaaatca 840

the

taggaaggct actatgattg gagaaccgtg gtgttgtata actgattaga ttctttcaaa 900

the

tatgattttc agttagaccg ctcattggtt caggatcagg gtcttttgca gttctcccaa 960

the

atattgcact aatttgtttg ccaaataatc atgcaaaact tacatgagtg gtggcattaa 1020

the

ctcttttatt caaatacatg gttgttcaat gatggtatta actcttctat tcaacaggct 1080

the

gtaaaaaatt aagtgaataa tccttgtccc gctacttaaa atctagtcaa gactcagatt 1140

the

ggaaaccatt ggggactagt aaagtttatg ggacttggac gtgtacatta catgcacatg 1200

the

cccatgatac attaacagga tggatgttct attctggaag gttaaatata atagttctta 1260

the

gaaagttaga gttttcagac cgatggtatg cttgcatata tttttagaag tatcaaatat 1320

the

gatgagagtt cttaaaagaa ttgggtttgt taactatgtt gagtgccttc tactatttt 1380

the

tttacaggtt tttatgttc atggtgatat actatatgca tgaataagtt acagttatgt 1440

the

aatatatatg ggcaaggatg tataagaagt tcatttgttt tctaaatttg tagcttaggc 1500

the

ttcttctgct gggctcttcc atgaagcaaa tgaagtttat aatgcacata ctaatgttca 1560

the

gtatattata ctgagaatca actttattct cagctgtata tacactagta tgccagccat 1620

the

tggtagctac ttgaaaggat ggtgaaacgc atatgattac ccatgatgag catgtgtgtt 1680

the

cccttttttt attagtgcta agctggaaat caataaatcc aagatattca tggagcatgc 1740

the

tacctgtgat gatgtgcatg agcatgctat aaatgtatca catggagaaa ctgcatcaac 1800

the

atcaaccagt catcaagatt tgcacagtga ttcagatgat tcacatcagg atgataggcc 1860

the

ttcaacaagc acacaaaccc catcaccaca gtcttcagca tcaacttcgc ccactgcata 1920

the

taacaccaga aatttatcct ttcctagaag agatagtatg tatggtcatg gaagaagtat 1980

the

ttggaattct ggtttgtgga tctcgtttga actggtcata tatgtagtac agattgtagc 2040

the

tgctattttc gtccttgtct tttcaagaga cgaacatccg catgcccctt tatttgcatg 2100

the

gataattggt tacacaattg gctgcattgc aagcattcct cttatttgtt ggcgctgtgc 2160

the

ccatcgaaac agaccttcgg aacaagaacc tgaacaacca cccgcagcct atcctaattt 2220

the

gacttcctct caatcatcag aaggacgcaa tcagcgtagc agtggtactg ttttgcattt 2280

the

tggatgtatc acaatttcgt gtccaaggta atttgtagtt ccatgttatt tttctatctt 2340

the

gaatttccta aagtcctgta tgtgaactcg tgtatgcagt ctcactccta tgcatttttt 2400

the

gaagaaactg ctatccattt gcatcttata aacatattta tgctttccaa taactgaaag 2460

the

atgcaacaaa taaactgata gttgaattga ttgaaactat caactctagg gcatcctaat 2520

the

tagttattt ttgatattcc attggatcac gcaggtacca cagtcctatg ttcttgagtt 2580

the

gagccttcta tttgtgcgta gtaccacgat atggattcta caatatattt ttactgcaat 2640

the

ttgttttttc tcagtcattc tggaagtgta gaagatatat atgctcttgt ttctttgacc 2700

the

taggaatttg gaacagttgt gttgacctct tcaaggtgta atttacaat gttcgctgca 2760

the

gatacctcat tacattggag gggaggatgt tccctttcta tatttgttgt gcttgacaaa 2820

the

agcattcaac attgtggagc gtaatgtgtc ctatttaact ggaattggtc ctgttgaaaa 2880

the

ttgtactagt ctactctaaa gtattaaaac tttataagta aatttgaaga aatgcgtgat 2940

the

gcgagatctt atagtactta gttttgtgct aaatttgata tggatgtatt aactgaggtg 3000

the

attatacatt acaggcctag catattggct tatcatttca agacagctgt agactgtttc 3060

the

tttgctgtat ggtttgttgt tggcaatgtg tggatttttg gtgggcacag cactttgtca 3120

the

gattctcagg aagctcccaa tatgtatagg tattttcttt cctatcattc atagcttttc 3180

the

tgaactcaat caactcatgc cttactttgt tgcctcttgc taggctatgc ttagcattcc 3240

the

ttgcacttag ttgtgttggg tatgctattc ccttcgtcat gtgtgcagcc atatgctgct 3300

the

gctttccatg cttaatttct cttctgcgcc ttcaagagga tttgggtcat actagaggag 3360

the

ctactcaaga actaattgat gcactgccaa ctacaaatt caagccaaaa cgaagcaaaa 3420

the

tgtgggttga ccatgcttca agctcagaga atcttagcga gggtggcatc ctgggcccag 3480

the

gaactaaaaa ggaaaggatt gtttcagctg aagatgctgt gagtatattt cacattttca 3540

the

tatcattttc atgtctgatg atacttgatt tgcaattagt attgagggggg tttccgtaaa 3600

the

acaagtattg atgggattcc ttgtatcgac cttcatgtac cttataattt agtaatcata 3660

the

actccattca caagtgaata cttaagcagc ctctgttatg cagtaatatg tactgtctta 3720

the

gccttatctt attttggaat atatttaaca aaaggtctag ctcgtgatga tgcttttaca 3780

the

catctttttc agataacaat gagacttccc ttttttcctc agtgaaacat atagtgttct 3840

the

aggtaaatat tcattaacaa aagtgtttta gggaaatatt ctgctttatg agaaatagtt 3900

the

ttgtttatat tatactacag tatctttttt ctcgtgtatc ttcaaacagt tgtagttaca 3960

the

ctcgctttca taagtccttt tctaaattcc attcatttcc ttcagtaaca tgggacctct 4020

the

tggaattttc caagttacct gacttactgt ctggattatt atttttgtag ctcattcaat 4080

the

ttgccattac taacttgaaa ccagggttct cgaaataatg ttatagttgt tttagtttga 4140

the

ttcataagtc ataacatata acttgtcaac atctattgat atctgcaggt gtgctgcatc 4200

the

tgtcttacta agtacggaga tgatgatgag ctccgtgagc ttccttgcac ccacttcttt 4260

the

catgtgcaat gtgtcgataa atggctcaag ataaatgcag tgtgcccact ctgcaagacc 4320

the

gagattgggg gtgtggttcg atcatttttt ggcttgccct ttggtcgccg acgtgttgat 4380

the

aggatggcag gaagaggtat agctagctcg agattcactg tatagaacac gtcttcttct 4440

the

ctagcatgtt tgcttgtttc atctgctcat atgcacataa aagacgtgct catggattgt 4500

the

agtttgttga tttgcaatga aagcgataat ctgctttcat cacccttgag ttcaccaaag 4560

the

tgatgacaga aaagtggaga cctgatgctt gcagtgacaa gtttctgcag tacagtagaa 4620

the

acataagtat attctgatgt aacatttgat gtcaagattg taaataaaga gcacaaagtt 4680

the

cacttcgggg gtgtatatct gcatgtgtat gggaaaggaa agcctaatta gttagtaact 4740

the

ttgtgggcat tttatgtgt gcaatcattg atcttgtttt tccc 4784

the

the

<210> 2

<211> 1245

<212> DNA

<213> Rice (Oryza sativa L ssp. indica)

the

the

<220>

<221> CDS

<222> (1)..(1245)

the

<400> 2

atg gag cat gct acc tgt gat gat gtg cat gag cat gct ata aat gta 48

Met Glu His Ala Thr Cys Asp Asp Val His Glu His Ala Ile Asn Val

1 5 10 15 15

the

tca cat gga gaa act gca tca aca tca acc agt cat caa gat ttg cac 96

Ser His Gly Glu Thr Ala Ser Thr Ser Ser Thr Ser His Gln Asp Leu His

20 25 30 30

the

agt gat tca gat gat tca cat cag gat gat agg cct tca aca agc aca 144

Ser Asp Ser Asp Asp Ser His Gln Asp Asp Arg Pro Ser Thr Ser Thr

35 40 45 45

the

caa acc cca tca cca cag tct tca gca tca act tcg ccc act gca tat 192

Gln Thr Pro Ser Pro Gln Ser Ser Ala Ser Thr Ser Pro Thr Ala Tyr

50 55 60 60

the

aac acc aga aat tta tcc ttt cct aga aga gat agt atg tat ggt cat 240

Asn Thr Arg Asn Leu Ser Phe Pro Arg Arg Asp Ser Met Tyr Gly His

65 70 75 80 80

the

gga aga agt att tgg aat tct ggt ttg tgg atc tcg ttt gaa ctg gtc 288

Gly Arg Ser Ile Trp Asn Ser Gly Leu Trp Ile Ser Phe Glu Leu Val

85 90 95 95

the

ata tat gta gta cag att gta gct gct att ttc gtc ctt gtc ttt tca 336

Ile Tyr Val Val Gln Ile Val Ala Ala Ile Phe Val Leu Val Phe Ser

100 105 110

the

aga gac gaa cat ccg cat gcc cct tta ttt gca tgg ata att ggt tac 384

Arg Asp Glu His Pro His Ala Pro Leu Phe Ala Trp Ile Ile Gly Tyr

115 120 125 125

the

aca att ggc tgc att gca agc att cct ctt att tgt tgg cgc tgt gcc 432

Thr Ile Gly Cys Ile Ala Ser Ile Pro Leu Ile Cys Trp Arg Cys Ala

130 135 140 140

the

cat cga aac aga cct tcg gaa caa gaa cct gaa caa cca ccc gca gcc 480

His Arg Asn Arg Pro Ser Glu Gln Glu Pro Glu Gln Pro Pro Ala Ala

145 150 155 160

the

tat cct aat ttg act tcc tct caa tca tca gaa gga cgc aat cag cgt 528

Tyr Pro Asn Leu Thr Ser Ser Gln Ser Ser Glu Gly Arg Asn Gln Arg

165 170 175

the

agc agt ggt act gtt ttg cat ttt gga tgt atc aca att tcg tgt cca 576

Ser Ser Gly Thr Val Leu His Phe Gly Cys Ile Thr Ile Ser Cys Pro

180 185 190

the

agg cct agcata a ttg gct tat cat ttc aag aca gct gta gac tgt ttc 624

Arg Pro Ser Ile Leu Ala Tyr His Phe Lys Thr Ala Val Asp Cys Phe

195 200 205 205

the

ttt gct gta tgg ttt gtt gtt ggc aat gtg tgg att ttt ggt ggg cac 672

Phe Ala Val Trp Phe Val Val Gly Asn Val Trp Ile Phe Gly Gly His

210 215 220 220

the

agc act ttg tca gat tct cag gaa gct ccc aat atg tat agg cta tgc 720

Ser Thr Leu Ser Asp Ser Gln Glu Ala Pro Asn Met Tyr Arg Leu Cys

225 230 235 240

the

tta gca ttc ctt gca ctt agt tgt gtt ggg tat gct att ccc ttc gtc 768

Leu Ala Phe Leu Ala Leu Ser Cys Val Gly Tyr Ala Ile Pro Phe Val

245 250 255

the

atg tgt gca gcc ata tgc tgc tgc ttt cca tgc tta att tct ctt ctg 816

Met Cys Ala Ala Ile Cys Cys Cys Phe Pro Cys Leu Ile Ser Leu Leu

260 265 270

the

cgc ctt caa gag gat ttg ggt cat act aga gga gct act caa gaa cta 864

Arg Leu Gln Glu Asp Leu Gly His Thr Arg Gly Ala Thr Gln Glu Leu

275 280 285 285

the

att gat gca ctg cca acc tac aaa ttc aag cca aaa cga agc aaa atg 912

Ile Asp Ala Leu Pro Thr Tyr Lys Phe Lys Pro Lys Arg Ser Lys Met

290 295 300

the

tgg gtt gac cat gct tca agc tca gag aat ctt agc gag ggt ggc atc 960

Trp Val Asp His Ala Ser Ser Ser Ser Glu Asn Leu Ser Glu Gly Gly Ile

305 310 315 320

the

ctg ggc cca gga act aaa aag gaa agg att gtt tca gct gaa gat gct 1008

Leu Gly Pro Gly Thr Lys Lys Glu Arg Ile Val Ser Ala Glu Asp Ala

325 330 335

the

gtg tgc tgc atc tgt ctt act aag tac gga gat gat gat gag ctc cgt 1056

Val Cys Cys Ile Cys Leu Thr Lys Tyr Gly Asp Asp Asp Glu Leu Arg

340 345 350

the

gag ctt cct tgc acc cac ttc ttt cat gtg caa tgt gtc gat aaa tgg 1104

Glu Leu Pro Cys Thr His Phe Phe His Val Gln Cys Val Asp Lys Trp

355 360 365 365

the

ctc aag ata aat gca gtg tgc cca ctc tgc aag acc gag att ggg ggt 1152

Leu Lys Ile Asn Ala Val Cys Pro Leu Cys Lys Thr Glu Ile Gly Gly

370 375 380 370 375 380

the

gtg gtt cga tca ttt ttt ggc ttg ccc ttt ggt cgc cga cgt gtt gat 1200

Val Val Arg Ser Phe Phe Gly Leu Pro Phe Gly Arg Arg Arg Val Asp

385 390 395 400

the

agg atg gca gga aga ggt ata gct agc tcg aga ttc act gta tag 1245

Arg Met Ala Gly Arg Gly Ile Ala Ser Ser Arg Phe Thr Val

405 410 410

the

the

<210> 3

<211> 414

<212> PRT

<213> Rice (Oryza sativa L ssp. indica)

the

<400> 3

the

Met Glu His Ala Thr Cys Asp Asp Val His Glu His Ala Ile Asn Val

1 5 10 15

the

the

Ser His Gly Glu Thr Ala Ser Thr Ser Ser Thr Ser His Gln Asp Leu His

20 25 30

the

the

Ser Asp Ser Asp Asp Ser His Gln Asp Asp Arg Pro Ser Thr Ser Thr

35 40 45 45

the

the

Gln Thr Pro Ser Pro Gln Ser Ser Ala Ser Thr Ser Pro Thr Ala Tyr

50 55 60 60

the

the

Asn Thr Arg Asn Leu Ser Phe Pro Arg Arg Asp Ser Met Tyr Gly His

65 70 75 80

the

the

Gly Arg Ser Ile Trp Asn Ser Gly Leu Trp Ile Ser Phe Glu Leu Val

85 90 95

the

the

Ile Tyr Val Val Gln Ile Val Ala Ala Ile Phe Val Leu Val Phe Ser

100 105 110

the

the

Arg Asp Glu His Pro His Ala Pro Leu Phe Ala Trp Ile Ile Gly Tyr

115 120 125

the

the

Thr Ile Gly Cys Ile Ala Ser Ile Pro Leu Ile Cys Trp Arg Cys Ala

130 135 140

the

the

His Arg Asn Arg Pro Ser Glu Gln Glu Pro Glu Gln Pro Pro Ala Ala

145 150 155 160

the

the

Tyr Pro Asn Leu Thr Ser Ser Gln Ser Ser Glu Gly Arg Asn Gln Arg

165 170 175

the

the

Ser Ser Gly Thr Val Leu His Phe Gly Cys Ile Thr Ile Ser Cys Pro

180 185 190

the

the

Arg Pro Ser Ile Leu Ala Tyr His Phe Lys Thr Ala Val Asp Cys Phe

195 200 205

the

the

Phe Ala Val Trp Phe Val Val Gly Asn Val Trp Ile Phe Gly Gly His

210 215 220

the

the

Ser Thr Leu Ser Asp Ser Gln Glu Ala Pro Asn Met Tyr Arg Leu Cys

225 230 235 240

the

the

Leu Ala Phe Leu Ala Leu Ser Cys Val Gly Tyr Ala Ile Pro Phe Val

245 250 255

the

the

Met Cys Ala Ala Ile Cys Cys Cys Phe Pro Cys Leu Ile Ser Leu Leu

260 265 270

the

the

Arg Leu Gln Glu Asp Leu Gly His Thr Arg Gly Ala Thr Gln Glu Leu

275 280 285

the

the

Ile Asp Ala Leu Pro Thr Tyr Lys Phe Lys Pro Lys Arg Ser Lys Met

290 295 300

the

the

Trp Val Asp His Ala Ser Ser Ser Glu Asn Leu Ser Glu Gly Gly Ile

305 310 315 320

the

the

Leu Gly Pro Gly Thr Lys Lys Glu Arg Ile Val Ser Ala Glu Asp Ala

325 330 335

the

the

Val Cys Cys Ile Cys Leu Thr Lys Tyr Gly Asp Asp Asp Glu Leu Arg

340 345 350

the

the

Glu Leu Pro Cys Thr His Phe Phe His Val Gln Cys Val Asp Lys Trp

355 360 365

the

the

Leu Lys Ile Asn Ala Val Cys Pro Leu Cys Lys Thr Glu Ile Gly Gly

370 375 380

the

the

Val Val Arg Ser Phe Phe Gly Leu Pro Phe Gly Arg Arg Arg Val Asp

385 390 395 400

the

the

Arg Met Ala Gly Arg Gly Ile Ala Ser Ser Arg Phe Thr Val

405 410

the

the

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 4

gtttgcgaca ttggagcctt c 21

the

the

<210> 5

<211> 23

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 5

aatgcttggg tatgctaggt gaa 23

the

the

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 6

tcctcggaga tgtttgacct tg 22

the

the

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 7

cagaaggtgt acgcaactct tgt 23

the

the

<210> 8

<211> 19

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 8

ccatccaaac acgccctaa 19

the

the

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 9

attgcccctt gctatggt 18

the

the

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 10

ttcgtggatg gagggagtac 20

the

the

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

the

<220>

<223> Primer

the

<400> 11

rgcgtttgta ggagtgccac 20

Claims (10)

1. a peptide species, it is following arbitrary for being selected from:

A) polypeptide that comprises the aminoacid sequence as shown in SEQ ID NO.3;

B) be included in the aminoacid sequence as shown in SEQ ID NO.3 or through lacking, replace, insert the aminoacid sequence of one or more amino acid gained, described polypeptide has high temperature resistance function;

C) there is the polypeptide of the aminoacid sequence shown in SEQ ID NO.3;

D) polypeptide of aminoacid sequence as shown in SEQ ID NO:3.

2. the polynucleotide sequence of this gene of gene is to be selected from following arbitrary polynucleotide sequence:

A) polynucleotide that comprise the nucleotide sequence as shown in SEQ ID NO.1;

B) comprising corresponding to the-1395 to-1406 Nucleotide in the nucleotide sequence as shown in SEQ ID NO.1 is that GGGGGGGGGGGG remains unchanged, in nucleotide sequence in addition, through lacking, replace, insert the polynucleotide sequence of one or more Nucleotide gained, its coded polypeptide has high temperature resistance function;

C) there are the polynucleotide of the nucleotide sequence shown in SEQ ID NO.1;

D) polynucleotide of nucleotide sequence as shown in SEQ ID NO:1;

E) the coding polynucleotide sequence of any polypeptide as claimed in claim 1.

3. comprise described in claim the carrier of 2 gene.

4. a host cell, it is characterized in that described cell comprises polypeptide claimed in claim 1, or gene claimed in claim 2, or the carrier described in claim 3, the preferred eukaryotic cells of described host cell, more preferably vegetable cell and yeast cell, the preferred grass cell of described vegetable cell, particularly preferably paddy rice ( oryza satival.) cell.

5. for screening, locate, separate the molecule marker of new nucleotide sequence, described mark is selected from:

A) insertion/deletion mark InDel5, this mark is at paddy rice the 9th karyomit(e) apart between galianconism end 9130-9150kb, and the amplified production of described mark has length polymorphism;

B) SNP mark, is called for short RBsp1407, and in sense high temperature paddy rice and high temperature resistance rice plant, based on polynucleotide sequence described in SEQ ID NO:1, the corresponding sequence of described mark is respectively TGT-3060ACA and TGG-3060ACA;

C) microsatellite DNA mark RM7364, this mark is at paddy rice the 9th karyomit(e) apart between galianconism end 9440-9450kb, and described mark has length polymorphism through the amplified production of primer.

6. be claimed in claim 5ly marked at screening, location, separate the application in anti-/ sense high temperature gene, wherein said high temperature preferably 42 ℃ or more than, 45 ℃ or more than, 48 ℃ or more than, 50 ℃ or more than.

7. plant breeding method, described method comprises the gene of application described in 2, or application rights requires the polypeptide described in 1, or carrier claimed in claim 3, or host cell claimed in claim 4, or mark described in claim 5 or 6.

8. make responsive to temperature type paddy rice revert to the method for non-responsive to temperature type paddy rice, the method comprises that making in responsive to temperature type rice genome Chromosome 9 by genetic manipulation is G corresponding to the coding mutation of the-3060 of SEQ ID NO:1.

9. the method for inducing paddy rice high temperature resistance phenotype, the method comprises making not possess in the rice genome Chromosome 9 of high temperature resistance phenotype by genetic manipulation inserts G corresponding to 5 of the-1394 of SEQ ID NO:1 ' end, wherein, described high temperature preferably 42 ℃ or more than, 45 ℃ or more than, 48 ℃ or more than, 50 ℃ or more than.

10. making responsive to temperature type paddy rice is the method for high temperature resisting type paddy rice, the method comprises that making in responsive to temperature type rice genome Chromosome 9 by genetic manipulation is G corresponding to the coding mutation of the-3060 of SEQ ID NO:1, and make in its responsive to temperature type rice genome Chromosome 95 ' end upstream of the-1394 insert a G, make gained nucleotide sequence identical with the-1395 to-1406 Nucleotide in the nucleotide sequence shown in SEQ ID NO.1, from-1395, comprise 12 continuous G away from initiator codon direction sequence, wherein, described high temperature preferably 42 ℃ or more than, 45 ℃ or more than, 48 ℃ or more than, 50 ℃ or more than.

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